Abstract

Cytoplasmic linker protein 170 (CLIP-170) is a prototype of the plus end-tracking proteins that regulate microtubule dynamics, but it is obscure how CLIP-170 recognizes the microtubule plus end and contributes to polymerization rescue. Crystallographic, NMR, and mutation studies of two tandem cytoskeleton-associated protein glycine-rich (CAP-Gly) domains of CLIP-170, CAP-Gly-1 and CAP-Gly-2, revealed positively charged basic grooves of both CAP-Gly domains for tubulin binding, whereas the CAP-Gly-2 domain possesses a more basic groove and directly binds the EExEEY/F motif of the C-terminal acidic-tail ends of alpha-tubulin. Notably, the p150(Glued) CAP-Gly domain that is furnished with a less positively charged surface only weakly interacts with the alpha-tubulin acidic tail. Mutation studies showed that this acidic sextette motif is the minimum region for CAP-Gly binding. The C-terminal zinc knuckle domains of CLIP-170 bind the basic groove to inhibit the binding to the acidic tails. These results provide a structural basis for the proposed CLIP-170 copolymerization with tubulin on the microtubule plus end. CLIP-170 strongly binds the acidic tails of EB1 as well as those of alpha-tubulins, indicating that EB1 localized at the plus end contributes to CLIP-170 recruitment to the plus end. We suggest that CLIP-170 stimulates microtubule polymerization and/or nucleation by neutralizing the negative charges of tubulins with the highly positive charges of the CLIP-170 CAP-Gly domains. Once CLIP-170 binds microtubule, the released zinc knuckle domain may serve to recruit dynein to the plus end by interacting with p150(Glued) and LIS1. Thus, our structures provide the structural basis for the specific dynein loading on the microtubule plus end.

Crystal structures of the CAP-Gly-1 and CAP-Gly-2 domains. (a) Domain organization of the CLIP-170 dimer. The N-terminal CAP-Gly domains directly bind MT/tubulin. This binding is autoinhibited by the C-terminal zinc knuckle domains, which also serve to recruit dynein by interacting with p150Glued and LIS1. (b) Ribbon drawings of the crystal structure of the CLIP-170 CAP-Gly-1 domain (Left), electrostatic potentials on the front molecular surface in the same molecular orientation (Center), and a 180° rotated image (Right). The region corresponding to the GKNDG motif is circled with yellow broken lines. (c) Same figures as in b but for the CLIP-170 CAP-Gly-2 domain. Electrostatic potentials of the front surfaces of the CAP-Gly domains of p150Glued (PDB ID, 2HL3) (d), CYLD (1IXD) (e), and F53.43 (1LPL) (f).

Interactions of CLIP-170 CAP-Gly domains with MT/tubulin and EB1. (a) Pull-down assays of tubulin with GST-CAP-Gly domains. To show clear visible tubulin bands, the applied amounts of proteins on the gel were 1:4:2 for CAP-Gly-12:CAP-Gly-1:CAP-Gly-2. (b) Pull-down assays of CAP-Gly domains with GST-EB1. (c) Pull-down assays of tubulin with GST–CAP-Gly-2 mutants. Wild-type and mutant GST–CAP-Gly-2 domains were immobilized on the resin and tubulin was used as an input. Labels for conserved lysines are bold and underlined. (d) Pull-down assays of EB1 with GST–CAP-Gly-2 mutants. (e) Cosedimentation experiments of CAP-Gly-2 domains with MT.

Solution structure of the CAP-Gly-2–peptide complex. (a) A best-fit superimposition of the final 20 simulated annealing structures of the CLIP-170 CAP-Gly-2 domain (residues 212–281) bound to the α3-tubulin peptide (447–451) with the lowest energies is displayed. The CAP-Gly-2 domain backbone (cyan) and selected side chains (blue) are depicted with the peptide (magenta). (b) Close-up view of the hydrogen bonds (dotted lines) formed in the complex along the arrow in a (Right). The side chain of Phe-236 is omitted for clarity. (c) Pull-down assay of mutated CAP-Gly-2 domains with the α3-tubulin peptide suggests a critical role for Asn-253 in peptide binding.

Comparison of CAP-Gly-binding sites for zinc knuckle domains, the tubulin and EB1 tail peptides. (a) Zinc knuckle-induced chemical shift changes mapped on the molecular surface of the CAP-Gly-2 domain. The α-tubulin peptide is shown for comparison. Residues whose signal intensities were strongly reduced, such that (Iref − Iper)/Iref > 0.95, are shown in red, >0.90 in yellow, and >0.85 in magenta, where Iref and Iper represent the signal intensity of the reference and perturbed spectrum, respectively. The orientation of the molecule is identical with that of Fig. 1. (b) The EB1 peptide (magenta lines) bound to the p150Glued CAP-Gly domain (gray ribbons with magenta side chains) in the crystal structure (2HL3). (c) The EB1 peptide bound to the p150Glued CAP-Gly domain is compared with the NMR structure of the α-tubulin peptide (cyan/green) bound to the CLIP-170 CAP-Gly-2 domain (blue) with 20 ensemble structures.